In clinical diagnostic settings, it has often been necessary to collect biological samples such a whole blood, red blood cell concentrates, platelet concentrates, leukocyte concentrates, tissue, bone marrow apirates, plasma, serum, cerebral spinal fluid, feces, urine, cultured cells, saliva, oral secretions, nasal secretions and the like in various containers or tubes for subsequent testing and analysis. Typically, the samples must then be transported to a different location, such as a laboratory, where personnel conduct specific tests on the samples.
Generally, a considerable amount of time elapses between obtaining the sample and analyzing it. A common and recurring problem, therefore, is the maintenance of the biological sample in a manner that prevents degradation, alteration or destruction of essential materials during the manipulations and/or preparations preceding analysis of the biological sample as a test specimen. All cells contain a number of proteases and, as long as the cells are intact, the proteases do not damage the cellular components. Once the cells are frozen, broken up or disrupted, however, protease enzymes begin to react on cellular proteins unspecifically, thereby quickly degrading the cellular proteins. In protein isolation, this can lead to degraded or fragmented proteins. This also can lead to a decline in protein levels during storage and transport of biological samples, thereby limiting the sensitivity of testing methods. The yield of the protein(s), therefore, can be drastically reduced and can endanger all further subsequent experiments such as, for example, protein quantification, 2-D gel plotting of proteins, drug development, Western blotting, reporter gene analysis, immunoprecipitations, epitope tagging, specific protein activity assays, etc.
It has long been recognized that the body fluid sample must be maintained and preserved during the manipulations and/or preparations preceding analysis as a test specimen. A number of different compositions have been developed for maintaining the stability of the cellular fraction or the non-cellular components of a test sample during the preparatory stages. These include, for example, the use of a water-soluble phosphate such as ATP and a chelating agent for the preservation of whole cells or cellular components (Publication EP 431385-A); the use of an acid, anti-bacterial drug, and fluorine compound in combination for stabilization of cells in urine (Japanese Patent Publication 05249104-A); the use of an aqueous solution of ethanol, aliphatic diol and polyethylene glycol for preserving cell or blood fluid components (Japanese Patent Publication 03295465-A); a reagent composition for biological assays that contains a reducible water-soluble trivalent cobalt complex, metallisable dye, and water-soluble polymer (U.S. Pat. No. 5,171,669); a stability control solution for determination of urobilinogen in urine samples (U.S. Pat. Nos. 4,677,075 and 4,703,013); the use of an aqueous solution containing phosphate buffer, albumin, glycine, and cysteine for stabilizing dehydrogenases (German Patent Publication DE2629808-A); a stabilizing composition comprising a buffer, alanine and mannitol for stabilization of freeze-dried protein compositions (Publication EP682944-A1); and the use of cationic poly-electrolyte and cyclic polyiol in aqueous solutions to stabilize proteins against denaturation on drying (U.S. Pat. No. 5,240,843). A number of stabilizing preparations have been commercially manufactured and sold, a notable example being the COMPLETE® protease inhibitor cocktail tablets for the inhibition of proteases during extractions from animal and plant tissues.
Despite the development and commercial availability of stabilizing preparations and compositions, the overwhelming majority of these are quite limited as to their usefulness and efficacy and do not lend themselves without major modification and alterations to specific clinical problems or a broad variety of different clinical and analytical settings. For example, many such compositions do not stabilize the proteases in the sample, thereby failing to preserve sensitive proteins in the biological samples.
In the area of blood collection, a common additive generally used in blood samples prior to centrifuging to separate the blood into cell layers is an anticoagulation additive. Typically, the anticoagulation additive is a buffered citrate or heparin in an aqueous solution. Blood collection tubes containing an anticoagulant are commercially manufactured and sold. An example of such a tube is disclosed in U.S. Pat. No. 5,667,963 to Smith et al.
Attempts have also been made to prevent the degradation of peptides by adding EDTA and/or proteolysis inhibitors to the sample. For example, U.S. Pat. No. 5,541,116 discloses the use of two protease inhibitors, anastatin and leupeptin, in combination with EDTA for stabilizing peptides in whole blood, serum or plasma samples. The patent, however, discloses stabilizing the samples by adding an adequate amount of the stabilizing combination to the samples themselves after they have been obtained or after thawing in situations where the sample had previously been frozen after collection thereof.
Additionally, it is known that in some laboratories studying proteomics, protease inhibitor cocktails are manually drawn into syringes and then injected into blood collection tubes. This procedure, however, is dangerous because of the likelihood of needle stick injuries. Moreover, the volumes of the protease inhibitor cocktails are subject to technician-to-technician variability because of the manual method of loading the protease inhibitor cocktails into the tubes.
The issue of industry standardization for a protease cocktail, i.e. a combination of protease inhibitors, is also significant. First of all, different companies and different researchers are introducing a spectrum of protease inhibitors to collected samples, particularly blood or a component thereof. The effectiveness and behavioral differences of the protease inhibitors and combinations thereof are, ostensibly, not well studied and, therefore, it is extremely difficult to correlate one researcher's analytical results to another researcher's results. For example, a 2-D gel could present a certain set of information for blood that has been introduced to a protease inhibitor cocktail supplied by Sigma-Aldrich Company and yet present a different set of information for blood introduced to a protease inhibitor cocktail supplied by Becton, Dickinson and Company or by Hoffmann-La Roche, Inc. In addition, most protease inhibitors range in toxicity from mildly toxic to highly toxic. Presently, there is no control and/or limit to potential exposure to these toxins, thereby jeopardizing the safety of healthcare and laboratory workers.
Measures must be taken to inhibit endogenous protease for the isolation and purification of proteins. Measures must also be taken leading to industry standardization. Accordingly, there is a continuing need in the industry for an improved method and a collection device for blood and other biological samples that preserve the proteins present in the biological samples.